Municipal Solid Waste Management through Anaecic Earthworm, Lampito Mauritti and their Role in Microbial Modification

By Ganesh Kumar Arumugam, Sekaran Ganesan*, Ramani Kandasamy, Ravindran Balasubramani and Prasad Rao Burusa
August 2004

The Authors are Research Scholars at the Department of Environmental Technology - Central Leather Research Institute, Adyar, in Chennai - India.

Abstract
A laboratory experiment was carried out for proper management of sewage sludge generated from municipal wastewater treatment plant. The experiment dealt with the stabilization, through the action of anaecic earthworm, Lampito mauritii, of mixtures containing Sewage sludge, Rice straw and Cow dung. The vermicomposting of mixtures were carried out for 70 days. The vermicomposting resulted in significant reduction of total organic carbon and increase in total kjeldhal nitrogen. The heterotrophic bacterial population increased throughout the experiment and enteric bacterial populations like Salmonella, Shigella and Escherichia species reduced dramatically and declined to nil on vermicomposting. There was no reoccurrence of these pathogens on further period. The mid gut analysis of the worm also proved the changes in microbial population during vermicomposting.

Key words: Vermicomposting, anaecic earthworm, heterotrophic bacteria, enteric pathogen, sewage sludge

Contents

▲ 1. Introduction

Effluent treatment plants (ETP) generally consist of physical, chemical and biological process to treat the wastewater discharged from industries or residential complexes. The dissolved organics in wastewater is generated as sludge mainly during primary and secondary treatment of municipal wastewater. During recent years the methodology of solid waste management has shifted from conventional disposal strategies such as incineration, landfill etc... to conversion of sludge into value added products (Liang et al., 2003). The solid wastes generated from agricultural activities include crop residues and animal excreta meet special attention for disposal or utilisation. The usage of these solid wastes by recycling can supply nutrients to vegetative plants and also improve soil physical conditions and its fertility (Mishra et al., 1989; Bhardwaj 1995 and Sudha Bansal et al., 2000). Some of these wastes can be directly added to the soil without any proper treatment as they do not possess any toxic pollutants (Lerch et al., 1992), but for wastes like biosolids from sewage plant much attention is required since they can produce toxicity and have depressive effects on metabolism of microorganisms (Ayuso et al., 1996). Further a wide variety of pathogenic microorganisms have been reported to be present in the sludge generated from treatment of municipal wastewater (Abdennaceur Hassen et al., 2001).

A literature review on solid waste management suggests that these solid wastes should be biocomposted before applying to soil in order to achieve biological transformation of the organic matter and to avoid potential risks of pathogens (Beloso et al., 1993; Gliotti et al., 1997 and Masciandaro et al., 2000). Biocomposting of solid wastes bring about stabilization of the organic matter and effectively reduces pathogen concentrations in sludges to very low levels (Burge et al., 1987; Millner et al., 1987). However, absolute removal of pathogens becomes difficult to achieve and many survives the composting process (Russ and Yanko, 1981; Sidhu et al., 2001). Incorporation of earthworm in biocomposting process has been considered to be an appropriate technology for biowaste management for producing nutrient enriched compost. Various investigators have established the viability of the technology using earthworms as a treatment system for different wastes (Hand et al., 1988; Harris et al., 1990; Logsdon 1994; Ndegwa et al., 2000; Elvira et al., 1997).

During the process of vermicomposting earthworms maintain aerobic conditions in the organic wastes through proper mixing and the biochemical process is enhanced by microbial decomposition of the substrate in the intestines and the earthworms convert a portion of the organic present in the wastes into worm biomass and excrete undigested/partially digested matter as worm cast (Benitez et al., 1999). Further earthworms also enhance soil microbial activity by improving the environment for microbes (Syers et al., 1979; Mulongoy et al., 1989). Much of the research on vermicomposting has been focused on the changes in the chemical parameters and informations available on the microbiota which determine the rate of vermicomposting are very little or perhaps nil. The focal theme of the present investigation covers the changes in bacterial populations during vermicomposting process, changes in composition of gut and composition of casting and their association with stabilization.

▲ 2. Materials and Methods

▲ 2.1 Solid Wastes and Earthworm

The sewage sludge was obtained from sewage treatment plant (STP) catering for a population of 5000 people in a residential complex. The sludge obtained from STP was dumped in an composting pit for 3 weeks, this was carried out to avoid exposure of earthworms to the initial temperature increase during the thermophilic phase of composting of organic content of sludge. The rice straw and cow dung were obtained from local agriculture practitioners. The adult earthworms, Lampito mauritii was used in this study as these species appeared to be predominant and accommodated well in the agricultural land where sewage water has been used for irrigation. The worms were collected by hand sorting and maintained in soil amended with cattle manure and nutrients at a temperature of ±26°C before the onset of the experiments.

▲ 2.2 Mixture of Substrates Used

Different mixture of substrates used were

  1. Mixture of Sewage Sludge and Rice straw, 3:1 (SR)
  2. Mixture of Sewage Sludge, Rice straw and Cow dung, 3:1:1 (SRC)
  3. Control sample containing Sewage Sludge alone (S)

The ingredients were mixed well and moistened with water up to 50% moisture contents. These treatment groups with 2 replicates were investigated. The substrate was placed into fibre bins and placed in natural environment in order to maintain favorable temperature for earthworms, as worms are highly sensitive to temperature fluctuations. Two hundred matured earthworms were introduced into composting bins for 20 kg substrate.

▲ 2.3 Growth Rate of Worms

The treatment groups of different composition were allowed to decompose for 70 days. Samples from the entire unit´s were taken out at 5th and 10th week of the study to measure the population of individuals. The worms were washed with distilled water and were weighed. After weighing the earthworms were replaced into the composting bins immediately to prevent the worms from desiccating.

▲ 2.4 Analysis of Vermicompost

  1. Chemical Parameters
    The sludge samples after vermicomposting process were taken at weekly intervals up to 10 weeks. The samples were oven dried, sieved to get equal size and analyzed for various parameters. The total kjeldhal nitrogen (TKN) and Total Organic Carbon (TOC) were determined in accordance with the procedures of standard methods.
     
  2. Bacterial Analysis in Compost
    The heterotrophic microbial populations in the composted samples were determined using pour plate method. The composted samples were homogenized and extracted in buffer solution and dilutions were made up to 10-8. Tryptic soya agar was used for plating and 1 ml aliquot of each diluted sample was poured and plated. Salmonella and Shigella species were enumerated using Deoxy cholate citrate Agar and Escherichia species using Eosin Methylene Blue agar. All the bacterial plating works was completed within 4 hours of sampling. The bacterial numbers were expressed as CFU g-1 (Colony Forming Units per gram) after 36 hours of incubation at 37°C.
     
  3. Gut analysis
    The worms were washed with sterile water to remove the surface microbial flora from its outer skin. The earthworms were then sacrificed by freezing and their whole body was dissected. The midgut region approximately (3-5 cm) was taken for analysis and all plating works for bacteriological analysis were carried out immediately.
     
  4. Casting Analysis
    Totally 5 earthworms were collected from all the experimental units and rinsed thoroughly in sterile water. The worms were then placed in petriplates containing tissue paper for 5 minutes. The fresh casts deposited were analyzed for different pathogenic bacterial count and total heterotrophic count.

▲ 3. Results

▲ 3.1 General Observation

After the initiation of the composting process, the average body weight of earthworm was monitored on 35th and 70th day. No mortality was observed throughout our experimental cycle and all earthworms were recovered alive during the process. In SR and SRC the body weight of earthworms was observed to increase considerably, against the decreasing body weight in control sample. The readily available nutrients in SR and SRC enhance the feeding activity of the worms, showing their increase in biomass whereas the depleted nutrient level in control resulted in the decrease of their biomass (Table 1).

Table 1 - Growth profile of L. mauritii on different substrates
Substrates Range of Temp.°C Range of Moisture % Weight of earthworm, gm/worm
0th day 35th day 70th day
Sludge + Rice Straw (SR) 19-27 40-50 0.65-0.85 0.75-1.2 0.85-1.5
Sludge + Rice Straw + Cow dung (SRC) 19-27 40-50 0.65-0.85 0.80-1.5 0.9-1.8
Sludge (S) 19-27 40-50 0.65-0.85 0.54-0.74 0.52-0.66

* All the values are expressed in grams

▲ 3.2 Chemical Analysis

  1. Total Organic Carbon (TOC)
    The TOC content of SR and SRC treatments decreased remarkably well indicating the rapid mineralization of the organic matter. The decrease in TOC content of SRC and SR treatment were 72% and 62% remarkably. However, the control sample established a poor reduction upto 17%. Interestingly the TOC reduction in SR and SRC treatments was continued upto 63rd day and thereafter the decrease in TOC content was not appreciable proving the stabilization of the compost (Figure 1).
     
  2. Total Kjeldahl Nitrogen (TKN)
    The TKN level significantly increased by the presence of earthworms in both the SR and SRC treatment, however in control also slight increase in TKN was monitored irrespective of its low organic composition. From day 35th both in SR and SRC treatment remarkable increase in TKN had been noticed (Figure 2)

▲ 3.3 Bacterial Analysis in Vermicomposting

  1. Salmonella, Shigella and Escherichia Species
    The salmonella population was about 13-18 x 104 CFU g-1 in all the treatment units at the onset of the vermicomposting. The population density began to decrease gradually from day 7. However, no remarkable decrease occurred in the control sample. In both SR and SRC, the salmonella population reached 1-2 x 10¹ CFU g-1 on 48th day and thereafter decreased to zero level (Figure 3). The Shigella population was reduced comparatively in higher rates than Salmonella in both SR and SRC treatment units. The average population density of Shigella was about 8-14 x 10³ CFU g-1 at the beginning of vermicomposting and reached the value 2-4 x 10¹ CFU g-1 on 14th day and it was zero in consecutive weeks in both SR and SRC, but in control experiments these populations were reduced only up to 1 x 10² CFU g-1 on the 70th day (Figure 4). The Escherichia count decreased from 11-15 x 104 CFU g-1 to 1-2 x 10¹ CFU g-1 on 42th day in SR and SRC units, but these populations were maintained throughout the vermicomposting period in the control experiments (Figure 5).
     
  2. Total Heterotrophic Aerobic Bacterial Count
    The population of total heterotrophs in the control was at a level of 10-16 x 104 CFU g-1 without any major increase or decrease in their count, whereas this population 11-18 x 104 in SR and SRC began to increase fastly from 21st day and reached maximum to level at 56th day and maintained in the same population density, from 63rd day no major increase in population were monitored (Figure 6).

▲ 3.4 Bacterial Analysis of Gut

During the initial stages of the vermicomposting (1st week) various pathogens like Salmonella, Shigella and faecal coliform bacteria were found to be dominant in the mid gut but during its subsequent weeks their level decreased, Bacillus and Pseudomonas species were found predominantly in the treatments of SR and SRC. But the Shigella, Salmonella and faecal coliform were found in control (Table 2).

Table 2 - Bacteriological analysis in Mid Gut
No. of Days 0 35 70
a. Salmonella sp. in gut
Sludge S 16-17x10³ 5-8x10³ 2-3x10³
SRC 15-18x10³ 1x10¹ Nil
SR 15-18x10³ 1x10¹ Nil
b. Shigella sp. in gut
Sludge 6-7x10² 7-9x10¹ 2-5x10¹
Sludge + Rice straw Cowdung 6-8x10² Nil Nil
Sludge + Rice straw 5-7x10² Nil Nil
c. Escherichia sp. in gut
Sludge 10-11x10² 7-8x10¹ 1-3x10¹
Sludge + Rice straw Cowdung 11-14x10² 1-2x10¹ Nil
Sludge + Rice straw 10-12x10² 1-3x10¹ Nil
d. Pseudomonas sp. in gut
Sludge 18-20x10² 20-22x10² 21-22x10²
Sludge + Rice straw Cowdung 18-19x10² 54-56x10² 36-40x10³
Sludge + Rice straw 19-20x10² 46-48x10² 26-28x10³
e. Bacillus sp. in gut
Sludge 8-10x10¹ 6-8x10¹ 2-3x10¹
Sludge + Rice straw Cowdung 12-16x10¹ 21-23x10³ 36-39x10³
Sludge + Rice straw 10-11x10¹ 16-18x10³ 27-31x10³

* All the values are expressed in CFU/ gm

▲ 3.5 Casting Analysis

Casting analysis doesn´t show much difference in bacterial count, and it resembles the gut bacterial count. The Shigella, Salmonella and Faecal coliform levels were decreased well and Bacillus sp. and Pseudomonas sp. levels were higher at the end of composting (Table 3).

Table 3 - Bacterial profile in casting
No. of Days 0 35 70
a. Salmonella in casting
Sludge (S) 27-29x104 16-18x10³ 12-14x10³
Sludge + Rice straw Cowdung (SRC) 35-39x104 1x10¹ Nil
Sludge + Rice straw (SR) 32-34x104 1x10¹ Nil
b. Shigella in casting
Sludge 10x13x10² 11-14x10¹ 3x10¹
Sludge + Rice straw Cowdung 10-13x10² Nil Nil
Sludge + Rice straw 10-14x10² Nil Nil
c. Faecal coliform in casting
Sludge 22-24x10² 9-16x10¹ 4-6x10¹
Sludge + Rice straw Cowdung 24-26x10² 1-2x10¹ Nil
Sludge + Rice straw 22-25x10² 1-2x10¹ Nil
d. Pseudomonassp. in casting
Sludge 19-20x10² 20-26x10² 24-26x10²
Sludge + Rice straw Cowdung 18-20x10² 66-68x10³ 92-94x10³
Sludge + Rice straw 18-20x10² 56-59x10³ 61-62x10³
e. Bacillus in casting
Sludge 14-15x10¹ 4-5x10¹ 2-4x10¹
Sludge + Rice straw Cowdung 14-16x10¹ 32-34x104 65-66x104
Sludge + Rice straw 14-15x10¹ 21-28x104 27-31x104

▲ 4. Discussion

During the process of vermicomposting earthworms transform waste constituents into a more useful vermicompost by grinding and digesting organic wastes with the help of aerobic and anaerobic microflora (Maboeta and Rensburg, 2003). The total organic carbon is metabolised into CO2. Various microflora present in the intestine of earthworm and in the waste are involved in the decomposition of organic carbon, moreover the gut enzymes play a dominant role in this process (Whiston and Seal 1988; Kavian and Ghatneker 1991). The total nitrogen conent of the vermicompost increased with time, due to rapid mineralization of organic nitrogenous compounds. Our results was supported by Sudha Bansal and Kapoor (2000) showing increasing nitrogen content as a result of carbon loss in vermicomposting of crop residues and cattle dung. The nitrogen content of the vermicompost depends on the initial nitrogen concentration of the waste, further enhanced decomposition results in lowering of C:N ratio (Talashilkar et al., 1999).

Moisture content of the waste increases the microbial activity in vermicomposting of sewage sludge. The treatment with lower moisture content requires a lag phase to initiate the heterotrophic microbial activity. Our results suggest that 40-50% moisture content as minimal requirement for microbial activity. Liang et al. (2003) proved 60-70% moisture content having maximal microbial activity and showed 50% moisture content as minimal requirement for rapid rise in microbial activity.

Although the earthworms enhance soil microbial activity by improving the environment favourable for microbes (Syers et al., 1979) the fate of microorganisms during the gut transit through earthworms is still controversial (Wolter and Scheu 1999). The gut analysis and casting analysis proved the removal of Salmonella, Shigella and faecal coliform, in 35 days. However, the Pseudomonas, cellulolytic bacillus sp. and heterotrophic bacterial population were increased at the end of vermicomposting period, indicating the selective nature of earthworms in the removal of microorganisms. This corroborates with the findings of the researchers proving that earthworms include microorganisms in their substrates as a food source and can digest them selectively (Edwards, 1988; Edwards and Bohlen, 1996); (Bohlen and Edwards, 1995).

The microbial population dynamics of whole vermicomposting process is very complex, so isolation of all bacterial species in the vermicompost was not completely accomplished. The most media used in the present investigation are selective medium and they do not support the growth of many organisms present in vermicompost.

▲ 5. Conclusions

These results presented in this paper indicate the way by which the biosolids can be composted along with rice straw and cattle dung. The decomposition process was enhanced by the presence of earthworm and aerobic heterotrophic population. Moreover the paper also precisely pinpoints the way by which the earthworm selectively inhibits the growth of bacterial pathogens present in domestic sewage sludge. The Rice straw and Cowdung facilitate the removal of organic carbon content of the sludge. Mineralisation of the nitrogenous compounds was also facilitated by the presence of Rice straw and Cowdung. Survival of pathogenic organism Salmonella, Shigella and Faecal coliform were observed to reduce to nil concentration after vermicomposting, proves this pathogens are eliminated as they enter in food chain of the earthworm. However, Pseudomonas and Bacillus sp. were considered to be microflora responsible for the reduction of pathogens and metabolisation of other organics in sludge and they were not vanished during vermicomposting.

▲ Figures

Acknowledgement

The authors are thankful to The Director, Central Leather Research Institute and Council of Scientific and Industrial Research (CSIR) India for encouraging and providing all the facilities to carryout the work.

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